prepared by tonnybite.KILIMANJARO CHRISTIAN MEDICAL UNIVERSITY COLLEGE

1. BASIC TECHNIQUES IN CLINICAL
CHEMISTRY LABORATORY

2. INTRODUCTION
• Computerization and the use of automated methods of
analysis allow a high degree of productivity and improve
the quality of services.
• The use of analyzers with integrated computer hardware
and software has made the job very easy for clinical
laboratories as it provides automatic process control and
data processing.
• Automation is the use of various control systems for
operating equipments and other applications with
minimum human intervention
• The use of automation in clinical laboratory enables to
perform many tests by analytical instruments with minute
use of an analyst.

3. • Understanding the basic principles of techniques and
the theory of instrumental analysis will provide a
working knowledge of instruments, application stop at
investigating in the clinical chemistry laboratory.
• The use of automated analyzer has many advantages
including:
– Reduction of workload
– Less time consumption per sample analysis
– More number of tests done in less time
– Use of minute amount of sample
– Decreased chances of human errors
– High accuracy and reproducibility

4. Types of Autoanalyzers
• Semi-autoanalyzer - In semi-autoanalyzer, pipetting of samples,
reagent mixing and incubation, etc. are done manually
• Batch Analyzer - In batch analyzer, one batch of a specific test is
performed at a time automatically by the analyzer. In these
instruments, reagent mixture is prepared and fed automatically,
and one reagent is stored in the machine for running a test.
• Random Access Autoanalyzers - In such analyzers, more than
one reagent is stored at a time. The samples are placed in the
machine, and it can perform any number of specific tests on
each sample
• During analysis on autoanalyzer, the samples react with
reagents and undergo chemical reactions under optimum
conditions.
• The measurements are processed automatically and output
signals are sent in the form of results

5. • A variety of technique shave been used in clinical
biochemistry Laboratories for sample testing
• Most fundamental methods include:
– Photometry
– Nephelometry
– Turbidimetry
– Fluorometry
– Electrophoresis
– Chromatography
– Mass spectrometry
– Biochip (Protein and DNA Chip/Array)
– Biosensor

6. Photometry
• Photometry is defined as the measurement of the
luminous intensity of light or the amount of luminous
light falling on a surface from such a source
• Many determinations made in the clinical laboratory are
based on radiant energy emitted, transmitted, absorbed,
or reflected under controlled conditions.
• The principles involved in such measurements are
considered in the following spectrophotometry,
nephelometry, turbidimetry, fluorometry.

7. COLORIMETRY
• The absorbance of a solution is the amount of light
absorbed by that solution.
• Beer’s Law state that the absorbance(A) varies
directly with the concentration of the solution(c) in
question. It is equals to the concentration of a
substance in solution multiplied by the length of the
path(b) that the light must pass through the
solution, multiplied by the molar absorptivity (a) of
the substance of interest
• A = abc

8. Internal parts of a spectrophotometer

9. • Then measure the absorbances of our
samples.
0.396 0.490 0.594 0.681 0.774
Abs = 0.51
Colourimetry

10. • In practice, a beam of light is passed through a
monochromator that provides selection of the desired region
of the spectrum to be used for measurements.
• The monochromatic light next passes through an absorbance
cell, where a portion of the radiant energy is absorbed,
depending on the nature and the concentration of the
solution.
• Any light not absorbed is transmitted to a photo-detector
which converts light energy to electrical energy that can be
registered on a galvanometer
• In operation, an opaque block is substituted for the cuvette,
so that no light reaches the photocell, and the meter is
adjusted to read 0%T (transmittance)
• Next, a cuvette containing a reagent blank is inserted and the
meter is adjusted to read 100%T (i.e., zero absorbance)

11. • Going back the other way
• Transmittance = IT/I0*100
• Absorbance = log10(I0/IT)
Converting Absorbance to Transmittance
• Abs = log(I0/IT)
• 10Abs = (I0/IT), inverting
• 10-Abs = (IT/I0),
• 100* 10-Abs = 100*(IT/I0),
• 102-Abs = %T
• 2 – log%T = Absorbance

12. TURBIDIMETRY
• Turbidity causes the decrease of the intensity of the
incident beam of light as it passes through a
solution of particles
• The measurement of this decrease in intensity of
the incident light beam that is caused by scattering,
reflectance and absorption of the light is called
turbidimetry

13. NEPHELOMETRY
• Nephelometry is defined as the detection of light
energy scattered or reflected toward a detector that
is not in the direct path of the transmitted light
• Common nephelometers measure scattered light at
right angles to the incident light

14. FLUOROMETRY
• The interaction of radiant energy with molecules or
particles in solution can result in either fluorescence or
light scattering
• Fluorescence occurs when a molecule absorbs light at one
wavelength and remits light at a longer wavelength
• Light scattering occurs when radiant energy passing
through a solution meets a molecule in an elastic collision,
which results in the light being dispersed in all directions
• Several automated fluorometric analyzers have been
developed for special applications in clinical laboratory,
because of their sensitivity, speed, simplicity, and reliability
• These include the flowcytometer, hematofluorometer,
fluorescence microscopy.

15. ELECTROPHORESIS
• Electrophoresis refers to the migration of charged solutes
or particles in a liquid or a porous supporting medium, such
as cellulose acetate sheets or agarose gel film, under the
influence of an electrical field
Definitions
• Anode: the positively charged electrode in electrophoresis
system.
• Cathode : negative electrode.
• Isoelectric point (pI)of a molecule: is the pH at which it has
no net charge and will not move in an electrical field
• Ampholyte or Zwitterion: is a molecule that can be either
positively or negatively charged e.g. : proteins, amino acids.

16. • Chemical species carrying an electrical charge
move either to the cathode or the anode in an
electrophoresis system, depending on the kind of
charge they carry
• In a solution more acid than the isoelectric point
of the solute, an ampholyte takes on a positive
charge and migrates toward the cathode
• In the reverse situation, it migrates toward the
anode

17. The rate of migration is dependent on factors
such as:
1) the net electrical charge of the molecule
2) the size and shape of the molecule
3) the electric field strength
4) the characteristics of the supporting medium
5) the operation temperature

18. Description of Electrophoresis
A schematic diagram of an electrophoresis system shows:
Two buffer boxes (l) with baffle plates contain the buffer
used in the process. In each buffer box is an electrode (2)
of either platinum or carbon, the polarity of which is fixed
by the mode of connection to the power supply.

19. Description of Electrophoresis
The electrophoresis support (3) on which separation take
place is in contact with buffer by means of wicks (strips) (4).
The entire device is covered (5) to minimize evaporation and
Protect the system. The direct current power supply may be
Either constant current or constant voltage or both

20. Automated Electrophoresis Systems
• Although electrophoresis was traditionally a
manual technique, it has been improved by
the introduction of prepackaged gels and
electronic systems that incorporated all the
necessary components and reagents to
perform the procedure easily.

21. TYPES OF ELECTROPHORESIS
Agarose Gel Electrophoresis
• It is a convenient method of electrophoresis that uses a
purified, essentially neutral fraction of agar called
agarose as a medium
• It has been successfully applied to the analysis of serum
proteins, hemoglobin variants, isoenzymes, lipoproteins
fractions and other substances
• The advantages of agarose gel include its lower affinity
for proteins and its native clarity after drying, which
permits excellent densitometric examination

22. Cellulose Acetate Electrophoresis(CAE)
• Cellulose acetate is a thermoplastic resin of
cellulose that is made by treating cellulose
with acetic anhydride to acetylate the
hydroxyl groups to form the raw material for
membranes contain about 80% air space in
the form of pockets
• An advantage of CAE is the speed of
separation (20 min-1h) and the ability to store
the transparent membranes for long periods

23. Cellulose Acetate Electrophoresis

24. Polyacrylamide Gel Electrophoresis (PAGE)
• In PAGE, individual gels are prepared in situ in glass tubes
by polymerizing a gel monomer and a cross-linking agent
with the aid of an appropriate catalyst
• The gel to be poured into the tubular-shaped
electrophoresis cell
• The spacer comb is thrown on top of the gel and left for
about 30 min for polymerization
• Protein with small concentration is mixed with the
coloured loading buffer and loaded into the well after
removal of a comb
• When constant voltage is switched on, the separation
takes place in the gel with the retardation of some
proteins due to the molecular sieve phenomenon

25. Polyacrylamide Gel Electrophoresis (PAGE)

26. Polyacrylamide Gel Electrophoresis (PAGE)

27. Isoelectric Focusing (IEF) Electrophoresis
• Amphoteric compounds such as proteins, can
be separated by virtue of migration in a
medium possessing a stable pH gradient using
isoelectric focusing electrophoresis(IEF)
• The protein moves to a zone in the medium
where the pH is equal to its isoelectric point
(pI)
• At this pH, the charge becomes zero and
migration ceases.

28. Isoelectric Focusing (IEF) Electrophoresis

29. CHROMATOGRAPHY
• Chromatography is a physical method of separation in
which the components to be separated are distributed
between two phases: Stationary phase and Mobile phase
• The primary goal of the chromatographic process is to
separate a mixture into its individual components called
solutes or analytes
• A chromatographic separation requires a sample to be
introduced into a flowing stream of gas or liquid (mobile
phases) that passes through a layer or column containing
the stationary phase
• If the mobile phase is a gas, the technique is known as gas
chromatography (GC) or if a mobile phase is a liquid, it is
called liquid chromatography (LC)

30. • The stationary phase may be particles of a solid or gel or a
liquid
• As the mobile phase carries the sample through the
stationary phase, the solutes with lesser affinity for the
stationary phase remain in the mobile phase and travel
faster and separate from those that have a greater affinity
for it
• Separation mechanisms include adsorption, affinity, ion
exchange, partition and steric exclusion chromatography
which describe the predominant chemical or physical
mechanisms used to separate solutes

31. Gel-Filtration Chromatography
• It is also known as steric exclusion chromatography,
gel-permeation, size exclusion, molecular exclusion,
molecular sieve chromatography and separate
solutes on the basis of their molecular size
• A variety of materials have been used as stationary
phases including crosslink dextran (Sephadex),
polyacrylamide (Bio-Gel), agarose (Sepharose)
• Molecules too large to enter the pores remain
exclusively in the mobile phase and rapidly are eluted
from the column
• Molecules that are intermediates in size (and Small
molecules) have access to various fractions of the
pore volume and elude slowly

32. Gel-Filtration Chromatography

33. • Gel-filtration chromatography has been used in
the clinical laboratory to ：
1. To determine molecular weights of macromolecules
2. To remove low-molecular-weight salts or buffer ions
from protein solutions.
Gel-Filtration Chromatography

34. Ion-exchange chromatography
• In ion-exchange chromatography, solutes in a sample
are separated by their differences in sign and
magnitude of ionic charge
• In practice, ionic analytes are selectively eluted from
ion-exchange resins by varying the pH and/or ionic
strength of the mobile phase.
• Anion-exchange resins are characterized by the
presence of strongly basic quaternary
amines(triethylamino-ethyl groups) or weakly basic
groups (aminoethyl, diethylaminoethyl) which can bear
a positive charge.
• Ion- exchange chromatography is widely used to
separate and remove inorganic ion from aqueous
mixtures